The present invention relates to a novel microorganism, novel enzymes and a novel process for producing the novel enzymes. More specifically, it relates to a novel microorganism belonging to the genus Plesiomonas and having ability to produce maltose phosphorylase and trehalose phosphorylase required for the enzymatic production of trehalose and novel maltose phosphorylase and trehalose phosphorylase obtainable from the microorganism as well as a process for producing the enzymes.
The present invention further relates to a novel process for enzymatically producing trehalose (O-.alpha.-D-glucopyranosyl-(1.fwdarw.1)-D-glucopyranoside).
More precisely, the present invention relates to a process for producing trehalose by utilizing the novel maltose phosphorylase and the novel trehalose phosphorylase derived from the novel microorganism belonging to the genus Plesiomonas.
Trehalose is a material that is expected to be used for various uses such as in pharmaceuticals, cosmetics and foodstuffs. Therefore, various attempts have been made to date to industrially produce trehalose. Those attempts can be roughly classified into three approaches.
One of those is extracting trehalose from microorganisms having ability to accumulate trehalose in their cells (J. Am. Chem. Soc., 72, p2059 (1950); German Patent No. 266584; Japanese Patent Un-examined Publication (KOKAI, hereinafter referred to as "JP-A") 3-130084; JP-A-5-91890; JP-A-5-184353; and JP-A-5-292986). This method comprises steps of culturing microorganisms, isolating the microorganisms, extracting trehalose from the microorganisms, purifying and crystallizing the extracted trehalose, and hence the process steps are very complex. In addition, productivity of trehalose is lower than other processes and a large amount of microbial extraction residue is remained as waste. Therefore, this method cannot be considered an economically efficient process.
For another approach, microorganisms extracellularly secreting trehalose (into their culture media) have been screened and fermentation methods where microorganisms belonging to the genus Brevibacterium, the genus Corynebacterium and the like are cultured to produce trehalose by their extracellular secretion (into their culture media) of trehalose have been developed (JP-A-5-211882). However, also in this approach, production yield, i.e., accumulation amount of trehalose in culture media is not so high (about 3% (W/V)). Therefore, to produce a large amount of trehalose in an industrial scale by this approach, it is necessary to provide a fermentation tank of a large volume and a purification means sufficient for the tank volume and thus this method is not economically advantageous. Further, this method also requires the separation of the microorganisms in order to obtain purified trehalose and, in addition, it requires further complexed steps to eliminate impurities other than trehalose, which have been produced by the microorganisms, and culture medium components.
As a method for solving all of the above-described problems, enzymatic methods have been already developed. As such methods, reported are a process for producing trehalose where maltose phosphorylase derived from microorganisms (Maltose:orthophosphate .beta.-D-glucosyltransferase) and trehalose phosphorylase derived from algaes (.alpha., .alpha.-Trehalose:orthophosphate .beta.-D-glucosyltransferase) are allowed to act on maltose in the presence of phosphate (Japanese Patent No. 1513517; Agri. Biol. Chem., 49, p2113 (1985)) and a process for producing trehalose where sucrose phosphorylase derived from bacteria (Sucrose:orthophosphate .alpha.-D-glucosyltransferase) and trehalose phosphorylase derived from Basidiomycetes (.alpha., .alpha.-Trehalose:orthophosphate .alpha.-D-glucosyltransferase) are allowed to act on sucrose in the presence of phosphate (Abstracts of the Congress of the Agricultural Chemical Society of Japan (1994), 3Ra14).
It is reported that trehalose was produced from maltose or sucrose with a high yield of 60 to 70% by these methods. In addition, since the raw materials used in these methods are purified sugars with high purities, trehalose enzymatically produced may be easily purified and hence these methods are considered industrially advantageous as compared with other methods. However, also in this method, the enzymes used for this method, in particular, the trehalose phosphorylase, are derived from algaes or Basidiomycetes such as euglena and grifola, and therefore preparation of the enzymes from these sources is not only economically disadvantageous but also technically difficult. In addition, since optimum pH values of the obtained trehalose phosphorylase, sucrose phosphorylase and maltose phosphorylase used together therewith are quite different from one another, it is very difficult to control a pH value when they are used in combination. Moreover, thermal stability of these enzymes is quite poor and the trehalose production can be carried out only in a low temperature range, for example, 25 to 37.degree. C. and this may lead microbial contamination during the enzymatic reaction when the reaction is carried out in an open reaction tank. Therefore, this method requires strict contamination control to prevent secondary reactions caused by contamination and it may be a drawback of this process. Furthermore, these known enzymes do not permit use of a high concentration of the raw materials when they are used in combination because of their substrate concentration dependency. Therefore, this method also is not an economically efficient method.
With the background described above, it can be said that, if novel maltose phosphorylase and trehalose phosphorylase which are easy to be produced and purified and have high thermal stability and no substrate concentration dependency have been found and become available, it is possible to produce trehalose with high yield and high efficiency from maltose, which is easily available in a large amount.
Therefore, the first object of the present invention is to provide a novel microorganism capable of producing novel enzymes satisfying the various requirements described above, i.e., novel maltose phosphorylase and novel trehalose phosphorylase, with a high production efficiency.
The second object of the present invention is to provide novel maltose phosphorylase and novel trehalose phosphorylase which are easy to be produced and purified and have high thermal stabilities and no substrate concentration dependency.
The third object of the present invention is to provide a process for easily producing the two kinds of enzymes described above with a high yield by utilizing the microorganism mentioned above.
In addition, the forth object of the present invention is to provide a process for producing trehalose by utilizing the novel maltose phosphorylase and the novel trehalose phosphorylase mentioned above and using maltose as a substrate, wherein the enzymatic reactions are possible at a relatively high temperature and at a relatively high substrate concentration and pH value is easily adjusted.